Synonyms

Vaccinia virus B1R-related kinase 3; Vaccinia-related kinase 3; VRK-3

Historical Background

VRK3 is a member of the vaccinia-related kinase family (Manning et al. 2002), which comprises a group of three proteins, of which two, VRK1 and VRK2, are catalytically active. VRK3 has substitutions in key residues within its catalytic domain and is under some conditions a pseudokinase without kinase activity (Nichols and Traktman 2004). The biological effects of VRK3 are mediated by protein-protein interactions. VRK3 can deactivate mitogen-activated protein (MAP) kinase signaling in the nucleus by activating phosphatases such as VHR, which dephosphorylates extracellular signal-regulated protein kinase (ERK), and reduces its ability to activate transcription. VRK3 kinase activity can be induced by its interaction with BAF that modifies the conformation of the vrk3 n-terminal domain (Park et al. 2015).

VRK3 Gene Expression

The human VRK3 gene is located in chromosome 19q13.33, has 15 exons, and its mRNA is 1.9 kilobases in length (Nichols and Traktman 2004); and although there are several alternative messages, only one protein has been identified.

Murine VRK3 gene is expressed throughout murine embryonic hematopoietic development. Peak expression occurs in liver at days E11.5 to E12.5, followed by a drop of an order of magnitude between days E13.5 and E15.5. Expression levels recover again by day E17.5 in embryogenesis. In fetal peripheral blood lymphocytes expression is high at days E10.5 to E11.5, which is followed by a two- to threefold reduction. Expression levels then remain constant until the end of fetal development (Vega et al. 2003). In the adult mouse, VRK3 mRNA is expressed at similar levels in the liver, kidney, muscle, thymus, and bone marrow. Expression in the adult spleen is one order of magnitude lower (Vega et al. 2003). In an independent study, mouse VRK3 mRNA expression was detected at very high levels in kidney, testis, and small intestine. VRK3 protein was also detectable at significant levels in heart, kidney, and testis, and at lower levels in lung, stomach, liver, spleen, and skeletal muscle. However, the variations in protein expression were much less significant and did not correspond with the large variations in mRNA expression (Kang and Kim 2008).

In rat embryos, the highest VRK3 expression was detected between E15 and E17. However, this study examined whole-embryo expression only during days E13 through E17 (Kang and Kim 2008). VRK3 is highly upregulated in some rat tissues including kidney and testis, which correlates with reduced levels of phospho-ERK (Kang and Kim 2008). Intriguingly, it has been reported that ERK activity promotes the transcription of VRK3, suggesting the existence of a feedback inhibitory mechanism (Kang and Kim 2006; 2008).

VRK3 Protein Structure

The VRK3 protein has 474 aminoacids and 52.8 kDa (Fig. 1). The crystal structure of VRK3 reveals that despite the conservation of the kinase domain fold (PDB: 2JII), mainly in its surface, it presents several changes that render it catalytically inactive, but may function as a scaffold protein and it maintains the overall structure of the kinase domain (Scheeff et al. 2009). The residues previous to the activation loop (304–311) are different. Also the ATP binding site has an acidic residue (D175) and interferes with ATP; furthermore a loop with L262 sterically clashes with adenine. The inactivating substitutions occurred early in evolution and hinder binding to ATP (Scheeff et al. 2009). VRK3 also has differences in the activation loop, in which a helix is replaced by a kink (Scheeff et al. 2009). Human VRK3 is a pseudokinase and plays a scaffolding role in the nucleus where it recruits the VHR phosphatase (Kang and Kim 2006; 2008). However, the mechanism through which its interaction with VHR is regulated has not been identified. VRK3 is a nuclear protein and has a nuclear localization signal in its N-terminal region (Nichols and Traktman 2004). The N-terminal region of VRK3 has a regulatory role and is able to induce the activation of the VRK3 kinase activity by an unknown mechanisms(Park et al. 2015), but probably involves a conformational change that requires the N-terminal regulatory region (1–147) (Park et al. 2015), which was not included in the structure determination (Scheeff et al. 2009). How this conformational change in the N-terminal region is regulated is unknown.

VRK3, Fig. 1
figure 2325figure 2325

Structure of human VRK3. NLS, nuclear localization signal; ZincR, Zinc-ribbon domain. The pseudokinase domain becomes kinase-active depending on the folding of the N-terminal region. BAF (barrier autointegration factor) interact with the N-terminus of VRK3 and activates its kinase activity. VRK3 activates the VHR phosphatase that dephosphorylates and inactivates ERK

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Studies with GFP-hVRK3 fusion proteins revealed that the protein is located in the nucleus. VRK3 has a bipartite nuclear localization signal within the first 149 aminoacids (Nichols and Traktman 2004). No variant proteins have been identified in vivo (Nichols and Traktman 2004).

VRK3 Signaling

Unlike other vaccinia-related kinases, VRK3 does not have kinase activity. Although it contains a weakly conserved catalytic domain that is related to casein kinases, this domain has several substitutions in residues that are crucial for catalytic activity (Nichols and Traktman 2004). Indeed, its structure reveals that it has a poorly conserved catalytic site, but a highly conserved kinase fold (Scheeff et al. 2009). The human (h)VRK3 and mouse (m)VRK3 proteins are catalytically inactive (Nichols and Traktman 2004) and VRK3 does not bind ATP (Kang and Kim 2008; Scheeff et al. 2009). This VRK3 pseudokinase domain might become kinase-active depending on specific interaction partners, such as baf, which by altering the conformation of vrk3 enhances its activity, by opening the ATP binding, site which is blocked by a large hydrophobic side chain and become a substrate (Park et al. 2015).

However, VRK3 plays a scaffolding role in the nucleus (Kang and Kim 2006). Human VRK3, by its region 420–453, is able to directly interact with and enhance the activity of the phosphatase VHR. This interaction results in dephosphorylation of phospho-ERK1/2 and subsequent downregulation of ERK signaling in the nucleus (Kang and Kim 2006; 2008). Furthermore, in rat neurons treated with cisplatin, the expression levels of VRK3 and the DUSP6 phosphatase are selectively reduced, resulting in an increased and sustained accumulation of phospho-ERK in these cells (Gozdz et al. 2008). Activation of ERK by phorbol ester (PMA: phorbol-12-mirystate 13-acetate), an analog of diacylglycerol, induces an increase in VRK3 levels, thus establishing a negative feedback in MAPK signaling mediated by VRK3 (Kang and Kim 2006; 2008).

In an analysis of the kinome in response to EGFR signaling, VRK3 and VRK2 correlated with low levels of p-ERK (Komurov et al. 2010), which is consistent with its role in recruiting phosphatases and dephosphorylating associated proteins.

Human VRK3 also preferentially interacts with the GDP-bound form of the Ran small GTPase, but the functional significance of this interaction is not known (Sanz-Garcia et al. 2008) (Fig. 2).

VRK3, Fig. 2
figure 2326figure 2326

VRK3 nuclear signaling. Activation of VHR and phosphorylation of BAF by VRK3 (Modified from Kang (Kang and Kim 2006))

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VRK3 Phosphoryates BAF and Nuclear Envelope Dynamics

In addition to the VRK3 pseudokinase role as a scaffold protein, it has been shown that under certain situations VRK3 can also have catalytic activity. The activation of VRK3 kinase depends on the folding of its N-terminal regulatory domain (residues 1–147). Active VRK3 phosphorylates the nuclear envelope protein barrier to autointegration factor (BAF) on Ser4 (Park et al. 2015). VRK3 facilitates nuclear translocation of BAF and is required for cell proliferation (Kang and Kim 2006). Baf phosphorylated by VRK3 interacts with lamin and emerin, a lem protein, and contributes to nuclear envelope disassembly in mitosis and is also involved in DNA damage responses (Montes de Oca et al. 2009). VRK3 might facilitate dissociation of chromatin or nuclear envelope proteins to promote either replication or gene expression (Park et al. 2015).VRK3 is regulated in cell division and its highest level is reached in interphase (Park et al. 2015).

Summary

VRK3 is the most divergent member of the VRK family. It has no kinase activity due to several substitutions in key residues within its catalytic domain. VRK3 is mostly located within the nucleus and appears to play a scaffold role, where it downregulates MAPK signaling and reduces the level of p-ERK by recruiting the VHR phosphatase. In addition VRK3 specifically phosphorylates BAF in Ser4 and induces BAF translocation to cytoplasm. VRK3 expression is cell cycle regulated, reaching its highest levels in interphase.